Alveolar bone atrophy may occur due to trauma, malignant tumors, and periodontal disease (Albandar 1990, Schwartz-Arad & Levin 2004, Irinakis 2006). Restoring the lost bone is crucial for the rehabilitation of the patient's function, phonetics and aesthetic demands.
Nowadays, the methods available for vertical bone augmentation include: distraction osteogenesis, bone blocks (autologic/allogenic or xenogenic) and guided bone regeneration (GBR). These techniques are surgically complicated, unpredictable and some of them are also associated with significant morbidity (Chiapasco et al. 2006, Rothamel et al. 2009, Greenstein et al. 2009, Esposito et al. 2006, Rocchietta et al. 2008). The idea of using a physical barrier for guided bone regeneration (GBR) was first described in the early 80's by Nyman et al (Nyman et al. 1982a, Nyman et al. 1982b, Nyman et al. 1987, Pitaru et al. 1987). It was demonstrated that creating a critical size intra bony defect and covering the defect with mucoperiosteal flap will lead to healing of the defect, mostly with soft tissue; while when a barrier to separate the bone defect from the soft tissue is used, it allows for bone regeneration into the defect (Dahalin et al. 1990, Kostopoulos et al. 1994a, Kostopoulos et al. 1994b, Dahalin et al. 1988). The biological principle of GBR also includes the creation of a space between a rigid barrier and the underlying bone which prevents epithelial and fibroblastic cells migration from the soft tissue, thus enabling the slower moving bone-forming cells to migrate from the underlying bone to populate the space (Polimeni et al. 2005, Schenk et al. 1994). Another important role of the membrane is blood clot stabilization. It was demonstrated that supra-crestal bone defects that were treated with ePTFE membrane and had root conditioning with heparin, showed compromised periodontal healing and bone regeneration (Haney et al. 1993).
As of today, bone regeneration within the bony envelope (intra-bony) using GBR technique is a common and relatively predictable procedure (Kim et al. 2004). However, only few studies that attempted to grow bone extra cortically using C3B resulted with modest success (Majzoub et al. 1999, Min et al. 2007, Lundgren et al. 1995, Wikesjo et al. 2003, Lioubavina & Kostopoulos 1999).
Both the calvaria and the jaw bones are formed through the intra-membranous bone formation pathway (Verna et al. 2002). The process begins with the proliferation of mesenchymal cells and deposition of extracellular matrix. This newly formed tissue guides cell migration and angiogenesis. The combination of matrix components with new vessels forms provisional connective tissue that is later transformed into bone along blood vessels (Lang et al. 2003).
Mesenchymal stem cells (MSC) contribute to the maintenance of various tissues, especially bone, in adults. They can be isolated form bone marrow, placenta, umbilical cord blood, or from adipose tissue in adults (Pittenger et al. 1999, Secco et al. 2008, Zuk et al. 2001, Yen et al. 2005). The first to describe these cells was Friedenstein in 1966. MSC present several characteristics that assist to identify them: adherence to culture plates, fibroblast-like phenotype and proliferation potential. The osteogenic potential of these cells was also recognized (Friedenstein). Since that time, in-vivo and in-vitro studies showed that MSC can be transformed into mesenchymal tissues such as: bone, cartilage, adipose, muscle and tendon. The characterization of cultured MSC relies on combination of several antigens: CD105, CD146, CD90, CD73 and CD44; furthermore, the hematopoietic origin of the cells should be excluded (CD45, CD14 and CD34) (Baksh 2004, Geregory et al. 2005).
Several preclinical studies and a few clinical studies showed the efficacy of cultured MSC for bone reconstruction. In animal models these cells showed osteoinductive properties (i.e production of ectopic bone) and improved healing of bone defects (Petite et al. 2000, Bruder et al. 1998). In humans, few clinical reports (case reports) were published: Quarto et al. 2001 reported on callus formation 2 months after administration of autologous MSC seeded on hydroxyapatite scaffold into non-union long bone defects. The I.V. infusion of autologous MSC to children with osteogenesis imperfecta resulted in increase in their body length and bone mineral content (Horowits et al. 2002).
Several attempts to heal mandibular defect in animals using MSC have also been recently published. These studies reported that the addition of MSC to scaffold have statistically improved bone regeneration compared with scaffold alone (Steinhardt et al. 2008, Jafarian et al. 2008).
Although extensively studied, several difficulties concerning the use of bone marrow MSC still exist. The main problems are the invasive nature associate with the harvesting of MSC and the morbidity of the donor. Additionally some reports suggest age-dependent decline in the proliferation and the osteogenic differentiation of these cells (Zhou et al. 2008). Finally, the limited amount of MSC in bone marrow aspirates requires ex-vivo expansion to obtain sufficient cell number for transplantation (Banfi et al. 2000). Thus, extensive research to improve our laboratory techniques and control clinical trials is still needed in order to confirm the potential of MSC to induce new bone formation.
Blood-derived endothelial progenitor cells (EPC)—In 1997, Asahara et al. discovered the presence of bone marrow-derived circulating endothelial progenitor cells in adult peripheral blood and human umbilical cord blood (Ashara et al. 1997, Murohara et al. 2000) that participate in post-natal neovascularization (Takahashi et al. 1999). Furthermore, these cell lines were found to participate in angiogenesis, vascular repair, blood-flow recovery after tissue ischemia and vasculoprotection. The current clinical use of EPC is limited to treating ischemic tissue after acute myocardial infarct (Isner & Losordo 1999, Kalka et al. 2000). During skeletal development or bone healing processes the recruitment of EPC by vasculogenic/angiogenic molecules (e.g. VEGF, PIGF, erythropoietin) is crucial (Ferguson et al. 1999, Giannoudis et al. 2007). Studies that explored the effect of local delivery of BMP2 & VEGF loaded on scaffold on bone regeneration suggest a synergistic effect in this dual delivery system (Patel et al. 2008, Kempen et al. 2009). Dual release of BMP2 & VEGF from scaffold implanted into calvaria-bone defect in rats enhanced bone formation after 4 weeks compared to scaffold alone or scaffold with only one GF (Patel et al. 2008).
Successful regeneration of large bone defects might also benefit from concomitant stimulation of vascularization and osteogenesis. Hence, we suggest that EPC and MSC based therapy might enhance both processes because these cells are capable of participating in vasculogenesis, might induce mesenchymal progenitors to proliferate or differentiate into osteoblasts and possibly at the same time gain plasticity to differentiate themselves into bone forming cells (Bick et al. 2006).
Identification of EPC is rather controversial and complicated since three different populations of EPCs were identified:                1. CFU—Hill—these EPCs have weak proliferative and vasculogenic activities;        2. Circulating angiogenic cells—these cells are obtained from adherent peripheral blood mononuclear cells (PBMNC) and do not form colonies in culture. They are positive to: CD133, CD31, CD45 and VEGFR-2.        3. Late EPCs—these cells are derived from adherent PBMNC and form endothelial colonies after 3-4 week of culture. They are thought to play a major role in revascularization in adults; they are positive to CD34, CD144, VEGF-R2 but negative to CD133, CD45 and CD14 (Hirschi et al. 2008, Hur et al. 2004, Yoder et al. 2007).        
The participation of EPC in bone repair was reported by some researchers. Lee et al. and Cetrulo et al. showed the participation of EPC in distraction osteogenesis model (Lee et al. 2009, Cetrulo et al. 2005). The injection of human CD34 progenitor cells into non healing femoral fracture in nude rats enhanced bone healing compared with control group. The addition of VEGF antagonist to this model impaired not only angiogenesis but also osteogenesis and led the authors to suggest that these cells have paracrine effect on bone forming cells. (Lee 2008, Fuchs 2009).
Transplantation of autologeus EPC into critical size gap in sheep tibiae revealed full bridging at 3 months in 6 out of 7 E PC-transplanted defects while non or minimal new bone formation was observed radiographically in 8 sham-operated defects (Rozen et al. 2009). These authors also suggested that the effect of EPC is not limited to vasculogenesis but that they are also capable to transform into bone cells. When sheep EPC were sub-cultured under osteogenic conditions they changed their morphology and formed nodular aggregates (1-2 mm diameter) following 1-2 weeks incubation and stained positively by von Kossa (vK), alizarin red (AR) and osteocalcin immunohistochemistry, which are markers of osteoblastic differentiation (Bick et al. 2006).
To be able to use cell-based therapy in animal and human defects a suitable scaffold is required that will not only serve as a vehicle for cellular delivery, but will also have osteoconductive properties. Tricalcium phosphates (TCP) is a synthetic scaffold used in the clinics for the reconstruction of bone defects. These materials provide a mineral matrix phase similar to that found in bone tissue. Following application, TCP is resorbed by osteoclastic activity and replaced by newly formed bone. Additionally, TCP as a synthetic material does not pose the risk of transmitting pathogenic agents (such is the case with allographs and xenographs). It is also being resorbed more rapidly when compared with a xenograft. (Rojbani H, Nyan M, Ohya K, Kasugai S. Evaluation of the osteoconductivity of a-tricalcium phosphate, β-tricalcium phosphate, and hydroxyapatite combined with or without simvastatin in rat calvarial defect. J Biomed Mater Res A. 2011 Sep. 15; 98(4):488-98.
By combining TCP with GBR we achieved a mean height of 5.5±0.24 mm new hard tissue under the capsule, significantly higher than bone formed when capsules were filled with Bio-Oss collagen (p<0.001). Histological analysis revealed that TCP was partially resorbed and replaced by new bone that was continuous with the original calvaria. Residual TCP particles were surrounded by vascularized dense connective tissue.